ABSTRACT Since 1995, hatchery-produced juvenile oysters have been
planted on numerous natural oyster bars in Maryland in an effort to
restore degraded reefs. As part of the monitoring effort, 27 discrete
hatchery plantings spanning 10 y of restoration were sampled during late
summer and fall 2009. Oyster shell height, dry meat weight, shell
weight, and clump height all increased significantly with age. Perkinsus
marinus infections were low in all sampled populations, but increased
with age. These data enable estimates of growth and shell production
rates, and highlight the low prevalence of disease in restored Maryland
oyster populations. The longevity of these dense patches suggests that
local metapopulation restoration may provide substantial ecological
services. The trends presented in this study may provide valuable
insights for refining management tools, adapting ongoing restoration,
and improving population modeling efforts.

KEY WORDS: oyster, restoration, Maryland, Crassostrea, Perkinsus

INTRODUCTION

For more than 10 y, eastern oyster (Crassostrea virginica)
hatchery-produced seed (spat-on-shell) has been planted on small
sections of degraded natural oyster bars to restore oyster reefs in
Maryland's portion of Chesapeake Bay. Data gathered from these
discrete plantings are unique in that the sampled populations are of
known ages with little to no natural recruitment (Tarnowski 2010). In
addition, plantings were located in legally protected sanctuaries and
reserves (not open to annual harvest), although, subsequent to sampling,
reports of illegal harvesting at many sites were received. The
restoration effort presented a unique opportunity to study the size and
disease status of individual cohorts of oysters from 2 mo to 9 y of age.
The data reported here may be beneficial not only to oyster biologists
and restoration and resource managers, but also for use in predictive
modeling efforts.

Although the eastern oyster is important both ecologically and
economically along the east and Gulf coasts of North America, many
aspects of its biology and ecology are still poorly understood. For
instance, data concerning the density of C. virginica in most parts of
Chesapeake Bay are surprisingly absent from the literature. In addition,
little is known about geospatial stock-recruitment relationships between
parental broodstocks and local spatfall. Furthermore, Maryland and
Virginia populations of C. virginica suffer from 2 parasitic diseases
(Haplosporidium nelsoni-MSX and Perkinsus marinus-Dermo) that infect
oysters most virulently at higher salinities (Ford 1985, Andrews 1996,
Burreson & Ragone Cairo 1996). Effects of these diseases on
mortality and growth vary substantially through space and time, and have
varied in the upper Chesapeake among tributaries (Tarnowski 2010).

Juvenile eastern oysters (spat settled onto oyster shell) used to
restore oyster reefs were produced at the University of Maryland Center
for Environmental Science Horn Point Laboratory oyster hatchery in
Cambridge, MD, and planted by the Oyster Recovery Partnership. Planting
densities were targeted at 1-2 million/acre although some bars may have
received significantly less as a result of variations in production or
planting area.

Sixteen oyster reefs of various ages were sampled within
sanctuaries or managed reserves in the Maryland portion of Chesapeake
Bay (Fig. 1). Managed reserves are defined as areas that are closed to
fishing until a specific median size is reached (101 mm). No managed
reserves that had been previously opened were sampled. Restoration sites
with only 1 planting, or 2 plantings at least 4 y apart, were selected
so that distinct cohorts could be identified based on a size frequency
analysis. Many reefs had multiple geospatially discrete plantings. From
August to October 2009, divers haphazardly collected 50 oysters from
each restored reef/planting. If 2 cohorts were expected, 50 oysters of
each size class were collected. All oysters collected were the product
of restoration plantings of hatchery-produced oysters.

[FIGURE 1 OMITTED]

Oysters were returned to the laboratory and stored at 4[degrees]C
until processed. All live oysters within a sample were enumerated and
measured for shell height to the nearest millimeter. All live oysters
were shucked, and shell weights were measured to the nearest milligram.
Spat (juvenile oysters less than 1 y old) per shell, oysters per clump,
and boxes (articulated shells with no oyster tissue) were also tallied.
All samples were inspected for the presence of naturally recruited spat.

Oysters processed for dry meat weight (10 per sample) were shucked
and the wet tissue was blotted with a paper towel and weighed on a
digital scale. The tissue was then placed into a drying oven at
60[degrees]C for 72 h and subsequently weighed. Not all samples were
processed for dry meat weight.

Sampled oysters were typically found in clumps of 3 or more. Clump
height was measured by placing a clump on the laboratory bench and
measuring the distance from the laboratory bench surface to the highest
shell margin of the clump perpendicular to the laboratory bench. Large
clumps in 3-, 4-, and 5-y-old population samples were haphazardly
selected to be measured to represent a maximum clump height of those age
groups.

Thirty oysters were haphazardly selected from each sample and
processed for P. marinus diagnosis according to Ray's fluid
thioglycollate culture method (Ray 1952, Ray 1966), modified according
to Burreson (2009). Small portions of the rectum, gill, and mantle
tissue from each oyster were excised and placed in a test tube with 9.5
mL sterile thioglycollate media. Each test tube was inoculated with 0.5
mL of a penicillin/streptomycin mixture and 50 [micro]L of nystatin.
After 5-7 days of incubation at 26[degrees]C, the tissue samples were
removed from the culture media, coated with several drops of
Lugol's solution, macerated, and covered with a glass coverslip for
inspection under a compound microscope. Enlarged trophozoites were
counted for each tissue sample, and infection intensities were assigned
based on the number and density of trophozoites visible under 40 x
magnification. Prevalence was calculated as the percentage of oysters
infected, and level of infection intensity was scored (rare, 0.5; very
light, 1; light, l; light to moderate, 3; moderate, 3; moderate to
heavy, 5; heavy, 5; very heavy, 5). Weighted prevalence (WP) was
calculated as the mean infection intensity score of all the oysters
tested.

A total of 27 discrete populations from 16 oyster bars were
sampled, 10 of which were reported to have been impacted by illegal
harvest. Populations were considered "illegally harvested" by
virtue of any one of the following 3 criteria: (1) a Maryland Department
of Natural Resources citation or arrest on a specific bar, (2) a report
from cooperating watermen, or (3) a eyewitness account from laboratory
staff.

Live oysters were found at all sites. Divers reported patchy
distributions of dense clusters of oysters at many of the sites, and few
boxes were found at any of the study sites. Abundance, spatial
distribution, and survival were estimated by patent tong surveys and
will be reported elsewhere. Samples gathered in this study showed no
natural recruits (i.e., oysters smaller than the expected size range for
any given planted population).

Sizes at age are presented as shell height and dry meat weight
(Table 1). Oyster shell height increased rapidly during the first few
years, then more slowly after year 3 (Fig. 2). Oysters planted at 1-2 mm
shell height reached a mean shell height of 20.1 [+ or -] 6.65 mm (SD)
4-8 wk after planting, and mean shell heights typically reached market
size (75 mm) after 2 y. A natural log regression of shell height with
age was significant and fit the data well (Ln Regression, P < 0.0001,
[R.sup.2] = 0.81, n = 27). However, for many older populations where
illegal harvesting had been documented (Fig. 2, open points), mean shell
height may be underestimated. Thus, a natural log regression of shell
height with age was performed for data where no illegal harvesting was
documented (years 1 5), which correlated better and may more accurately
reflect natural oyster growth rates (Fig. 2 inset, P < 0.0001,
[R.sup.2] = 0.93, n = 17). Estimated mean shell height at age was higher
for these data.

[FIGURE 2 OMITTED]

Dry meat weight increased steadily with age (Ln Regression, P =
0.058, [R.sup.2] = 0.71; Fig. 3), such that mean spat dry meat weight
was 0.06 [+ or -] 0.04 g and increased over time to 2.53 [+ or -] 0.64 g
at 9 y of age. The low degree of significance observed in the dry meat
weight regression was partially a result of a single value more than 3 g
for a 4-y-old population at Shoal Creek (Fig. 3). This value was
confirmed and was not considered an error, but representative of the
high degree of variation in dry meat weight, and indicative of the
potential for tissues to grow quickly. A linear regression of the dry
meat weight data excluding illegally harvested populations (Fig. 3,
inset) was highly significant (P < 0.0001, [R.sup.2] = 0.98).

Disease prevalence was low and WP (the mean infection intensity
(see Burreson 2009)) values indicated that expected disease-related
mortality should have been low as the low box counts indicated. In
populations younger than 6 y of age, P. marinus prevalence was less than
40%; however, 7 to 9-y-old populations showed increased prevalence up to
90%. WP was less than 0.8 in oysters up to and including 6 y of age
(Fig. 4), whereas 7-y-old oysters had a WP of 1.7 and 9-y-old oysters
had a WP of 1.8. Weighted prevalence values above 3 are typically
associated with mortality in this region (Tarnowski 2010).

Although mortality was not quantified in this study, box counts
served as a crude estimate of oyster death. This is especially true
where oysters were collected in clumps, which serve as an informal
sampling unit. The observation of few boxes or scars within a clump of 3
10 oysters suggests low mortality and is corroborated by other
monitoring data. However, box counts should be regarded as a qualitative
estimate of mortality and are likely an underestimate (Mann et al.
2009b). Abundance estimates and long-term mortality will be estimated by
patent tongs surveys.

[FIGURE 4 OMITTED]

DISCUSSION

Oyster restoration in Maryland has produced several long-lived
patchy oyster reefs on previously degraded historical oyster bars. The
oysters living on these reefs were planted in areas of relatively low
salinity (mean salinities of <12 ppt) with the purpose of increasing
densities in areas with infrequent natural recruitment. These
populations acquired relatively low levels of P. marinus infections even
after many years, and the patchy reefs created complex benthic
structures and have shown remarkable community development (Rodney &
Paynter 2006). Although the data sets generated by this ongoing effort
may not constitute "successful" restoration per se, they may
be valuable in generating measures that could be incorporated into
larger assessments and predictive modeling.

[FIGURE 5 OMITTED]

Modeling studies for oyster restoration require accurate growth
rate and shell budget data (Baird & Ulanowicz 1989, Powell 1992,
Dekshenieks et al. 2000, Klinck et al. 2001, Cerco & Noel 2007,
Cerco & Tillman 2008, Tillman & Cerco 2009, North et al. 2010).
However, growth rate estimates used for management decisions in Maryland
have come largely from suboptimal sources such as floating tray culture
(Paynter & DiMichele 1989, Paynter & Burreson 1991) and stock
assessments (Jordan et al. 2002, Jordan & Coakley 2004). Also,
empirical data available in the literature on oyster shell production is
rare and based on the contribution of living oysters that contribute
their shells to the shell resource when they die. Powell and Mann
(Powell et al. 2006, Mann & Powell 2007, Powell & Klinck 2007)
argue that shell production rates may be limiting in Delaware Bay and
Chesapeake Bay. The results presented here provide empirical data for
shell weight with age for oysters up to 9 y old, and provide a
statistically robust equation for predicting such production rates in
other populations. The trends observed in this study suggest that the
growth rate and shell production of oysters in Maryland produce older
reefs that provide disproportionately larger ecological services than
their younger counterparts (discussed later and see Newell and Langdon
(1996)).

Growth Rates

Change in shell height and dry tissue weight with age represent
oyster growth rates. The growth curve of shell height with age showed a
remarkably good fit even though the data were collected from many
different locations, and many populations were impacted by illegal
harvesting ([R.sup.2] = 0.7630). Both curves were highly significant,
but note that 4 of the 5 populations older than 6 y fell below the
fitted line and were all reported to have been illegally harvested. The
growth curve generated for the populations without reports of illegal
harvesting (Fig. 2, inset) was a better fit ([R.sup.2] = 0.948).
Estimates of growth rates are important for modeling oyster production,
and these results will be especially valuable for predicting the growth
of oysters in low-salinity waters typical of many Maryland oyster bars.
The data show remarkable similarity to those generated by Coakley
(2004), who compared fished and nonfished oyster populations, indicating
that fishing reduces a population's mean shell height through the
preferential harvest of large oysters as the population grows into a
harvestable size range. Therefore, the size at age estimates for the
oldest ages in this study were likely underestimates. Results presented
here are also similar to those of Liddel (2008), who created a unique
von Bertalanffy growth equation for oysters in the bay based on
different populations, and to the growth estimates created for the
demographic model of a recent environmental impact statement on oyster
restoration in Chesapeake Bay (Volstad, et al. 2007). Thus, these
predictive exercises have been corroborated by the empirical data
presented here.

[FIGURE 6 OMITTED]

Dry tissue weight (grams dry weight (dw)) are an important
reference metric for many biological and ecological parameters. For
instance, filtration, clearance, and biodeposition rates in most bivalve
studies are reported by grams dw (Newell & Langdon 1996). Dry tissue
weight from the sampled populations approached a maximum of 2-3 g after
5 y (Fig. 3), although probably not in a linear fashion as the inset
shows. Again, large older oysters may contribute disproportionately to
important ecological functions like clearance and denitrification rates
(see the later section Ecological Functions; Table 2). Many reports use
1 g dw/oyster as an estimate of adult tissue weight, and therefore may
be underestimating mass-based estimates of such traits as fecundity or
filtration rate (discussed later).

Perkinsus marinus

Disease has been shown to affect oyster growth and fecundity
directly; however, disease prevalence in oyster populations varies
temporally with local environmental conditions, especially temperature
and salinity (Andrews 1996, Hoffmann et al. 2009). The current
conceptual theory of the P. marinus life cycle assumes a certain level
of subclinical, overwintering infection that is reactivated each year by
warming spring temperatures, and accumulates through time (Ragone Calvo
& Burreson 1994, Burreson & Ragone Calvo 1996). Because WP
values more than 3 are typically associated with mortality in this
region (Tarnowski 2010), the P. marinus infection levels in these
restored populations are generally nonlethal, even though the oysters
were up to 9 y old. Similarly, studies using triploid oysters in the
Patuxent River reported low levels of P. marinus infection both
initially and after seasonal intensification (Paynter et al. 2008,
Kingsley-Smith et al. 2009). In contrast, oysters deployed
experimentally in the Patuxent River by Albright et al. (2007) rapidly
contracted lethal levels of P. marinus infection at salinities
comparable with those observed at all sampling locations. These
disparate findings are indicative of the variable nature of P. marinus
epidemiology and infection rates among tributaries within the Maryland
portion of Chesapeake Bay. Restored oyster in the Severn River had lower
prevalence and WP values than nearby native oysters, whereas restored
oysters in the Chester and Choptank rivers showed disease levels similar
to or greater than those in nearby native oysters (Table 3). It is
important to note that during periods of oyster growth for the oldest
oysters reported here (1999 to 2009), salinities ranged widely at all
sites from 1.1-20.2 ppt (W. Romano, NOAA Chesapeake Bay Office, pers.
comm.), and included a nearly 4-y drought from 1999 to 2002. The WP
values presented in Figure 4, although likely a snapshot of a variable
annual trend, characterized the generally low levels of P. marinus
infection typical of many restored populations found at interim sampling
periods. Although boxes are typically thought of as indicators of recent
mortality, most of the oysters collected in this study were retrieved as
clumps, indicating minimal disturbance since planting (Fig. 5). Thus, we
expect many boxes may have been preserved longer than those on annually
fished bars, and the lack of mature fouling communities we observed on
the inside surfaces of these boxes, such as large barnacles, seemed to
bear this out. We would argue that the absence of significant numbers of
boxes in these sanctuaries and within clumps indicates low long-term
mortality.

production in portions of Chesapeake Bay or Delaware Bay. The data
gathered during this study may be a useful addition to the discussion of
shell budgets, especially with regard to the importance of long-lived
oysters. Because annual shell addition to the resource is based on
oyster mortality, that contribution is limited by the mean size of
oysters at the time of their death. In general, the mean shell weight of
oysters reported here were much larger than those used by Powell and
Klinck (2007) (Fig. 1), although the relationships between shell height
and shell weight in both populations were nearly identical. Thus, the
older populations of oysters in Maryland may contribute more shell mass
to the ecosystem, and therefore may play a relatively larger role in the
creation of reefs and maintaining and building reef height despite their
low mortality rates. These older communities of oysters are able to
survive for a variety of reasons, including the low prevalence of P.
marinus and the boring sponge Cliona celata, which is known to be
extremely destructive to oyster shell in higher salinity conditions such
as those found in Virginia and Delaware Bay (Pomponi & Meritt 1990).

Shell weight and clump height data from the 16 discrete plantings
suggest that shell production on reefs in Maryland would produce
significant deposits of calcium carbonate. Individual shell weight
increased to 100 g within 3 y (Fig. 5), suggesting that a target density
of 100 oysters/[m.sup.2] could yield 10 kg/[m.sup.2] or, 100,000 kg/ha.
Therefore, observed shell production rates, in addition to regular
robust recruitment, could substantially augment shell resources on any
given reef. Of course, shell contributions by long-lived oysters may be
mitigated by lower mortality rates over time, because the models
developed in the aforementioned studies require oyster mortality for
shell to be contributed to the "shell resource." Also, clump
height (Fig. 6) reached 10-12 cm within 4 y, suggesting oyster growth
rates might outpace sedimentation rates in many areas. Clump structures
remained intact for several years in areas undisturbed by destructive
forces, and contribute substantially to reef structure and complexity.
Furthermore, increases in shell-based structure and the overall
architecture of a reef probably increase faunal abundances (Harding
& Mann 2001, Rodney & Paynter 2006).

Ecological Functions

Many ecological contributions of oysters, including water
filtration, fecundity, and nutrient removal, have been directly related
to dry tissue weight (Riisgard 1988, Cox & Mann 1992, Choi et al.
1993, Newell et al. 2004). Using the data collected in this study,
estimates of several ecological contributions of different age oysters
can be calculated (Table 2). For instance, Riisgard (1988) postulated
that the filtration rate of an oyster was directly related to dry meat
weight. Applying those methods to various-age oysters it appears that
8-y-old oysters could filter 15 times more water per hour than a
2-mo-old spat, and 3 times more than a 2-y-old oyster (Table 2). Of
course, filtration rates are likely to vary with oyster health and
environmental changes (Powell et al. 1992), but the generalization may
still be accurate. Similarly, Choi et al. (1993) showed that fecundity
in West Bay, TX, oysters was directly related to dry meat weight. Their
formula suggests that an 8-y-old oyster could produce about 58 million
eggs per year (Table 2), nearly an order of magnitude more than 2-y-old
oysters. Finally, data from Newell et al. (2004) show that oysters
denitrify about 1.813 x [10.sup.-5] g N/L water filtered (Table 2). The
data presented in Table 2 suggest that reefs with higher densities of
small oysters might match the filtration and denitrification rates of
less dense reefs composed primarily of large oysters.

Restoration Challenges and Future Directions

Many studies have shown that restored reefs provide valuable
habitat for a wide variety of fauna, and the ecological benefits of
oysters in marine protected areas and/or sanctuaries are well documented
(Coen & Luckenbach 2000, Harding & Mann 2001, Luckenbach et al.
2005, Rodney & Paynter 2006, Powers et al. 2009). Unfortunately, we
know that many, possibly all, of the oyster populations sampled for this
study have been impacted by illegal harvest. The effects of illegal
harvesting are difficult to quantify, but likely include a reduction in
mean oyster size within a population, especially on older reefs, as well
as an overall reduction in oyster density. This makes the estimation of
natural and disease-related mortality increasingly complex. Illegal
oyster harvest is rapidly becoming epidemic in Maryland, with 124
citations issued from July 2008 to February 2010 (Maryland Department of
Natural Resources). This activity, paired with low natural recruitment
in Maryland, threatens the success of oyster restoration efforts.

A recent report of "unprecedented success" in restoring
oysters in the Great Wicomico River was based on a localized natural
recruitment event and the survival of a large portion of 1-y-old oysters
after shell plantings (Schulte et al. 2009). However, less than 10% of
the "restored" population in the Great Wicomico River in
Virginia was comprised of oysters older than 2 y ([greater than or equal
to] 70 mm). Given the paucity of large old oysters, that report calls
into question the definition of success in oyster restoration. Because
oyster survival and recruitment vary with salinity in Chesapeake Bay
(high recruitment and low long-term survival in high salinity, low
recruitment and high long-term survival in low salinity), a single
measure of success may not be appropriate to assess restoration efforts
baywide. Mann and Powell (2007) suggest sustainability (e.g., natural
recruitment must equal or be greater than annual mortality) should be a
requirement of successful oyster restoration. However, they also note
that the systemwide goal may be unobtainable, whereas local restoration
may prove more profitable for aquaculture and may provide local
ecological benefits (Mann & Powell 2007). This may be especially
true in Maryland's waters where disease-related mortalities are low
(Tarnowski 2010) and patch-specific densities reach more than 200
oysters/[m.sup.2]. Thus, the longevity of these dense patches suggests
that local metapopulation restoration may provide substantial ecological
services as one measure of success.

These data lead to several important conclusions. First, oysters
planted in the areas studied grew well enough to produce market-size
oysters in 2-3 y. These growth rates would likely support a vigorous
aquaculture industry. Second, the rates of P. marinus infection in the
oysters studied were low, suggesting that disease-related mortality
would not often threaten an aquaculture industry within the study area.
Third, the longevity of oysters in sanctuaries might substantially
increase the ecological value of local restoration efforts, at least in
terms of habitat creation and enhanced local biogeochemical processes.
These results suggest that oyster restoration efforts in Maryland could
result in significant success either through oyster production or local
reef ecosystem function.

The successes and challenges of oyster restoration in Maryland to
date suggest that localized efforts might provide the greatest
ecological and economic return. Restoration has historically been spread
across the bay in many different areas, diluting the effort in any
specific area. The scales at which restoration has been undertaken in
Maryland are insignificant compared with the scale of the area across
which they have been spread. Thus, any potential ecological signal from
the restoration effort has been lost in the noise of environmental
variation. However, if the efforts were to be concentrated, the
ecological signals might be detected and we might learn how best to
restore oysters locally in subestuaries and rivers. Understanding how to
maximize the ecosystem benefit of oysters at small scales will give us
the knowledge to attempt baywide management and restoration.

ACKNOWLEDGMENTS

Funding for the efforts described in this report was provided by
the Oyster Recovery Partnership, the National Oceanic and Atmospheric
Administration Chesapeake Bay Office, the Maryland Department of Natural
Resources, and the Army Corps of Engineers Baltimore District. Thanks to
Anthony Strube, Kennedy C. Paynter, and Grace Chon, who assisted with
sample collection, laboratory processing, and data management. The
authors also thank the dedicated hatchery crew at the University of
Maryland's Horn Point hatchery for their efforts in producing the
spat used in this study. The research vessel Callinectes was used to
collect the samples reported in this study. Thanks also to Charlie
Frentz, Stephan Abel, Eddie Waiters, and the field crew of the Oyster
Recovery Partnership for their help in coordinating and planting the
oysters associated with this study. Thanks to Michael Wilberg for his
critical review of the manuscript and to Chris Dungan for providing 2009
disease data.

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